Cellular homeostasis is regulated by cell renewal and apoptosis. Various apoptotic stimuli result in the recruitment of B cell lymphoma 2 (BCL-2) family proteins to mitochondria, the permeabilization of the outer mitochondrial membrane, the release of cytochrome c, and the activation of caspase, which are followed by processes leading to cell death. Formation of mitochondrial membrane pores is considered a point of no return in terms of cell life or death.
Bax is a 21 kD pro-apoptotic protein of 192 amino acids comprised of 9 alpha helices. Alpha helix 5, which is imbedded within the protein, is the most hydrophobic. Bax also has an extensive hydrophobic groove spanning one face of the protein and is comprised of alpha helices 2, 3, 4, and 5. When Bax is in its cytosolic, monomeric form, the amphipathic alpha 9 helix resides within the groove, and upon conformational change, the helix dissociates from the groove allowing for oligomerization. This conformational change can allow for translocation of Bax to the mitochondrial membrane due to the exposure of the C-terminal helix and relaxation of the bulk of the protein releasing the alpha 5-alpha 6 helices.
Bax was first identified as a protein that associates with and suppresses the anti-apoptotic protein, Bcl-2. As a member of the pro-apoptotic sub-family of Bcl-2 proteins, Bax retains the characteristic helical packing and hydrophobic groove that are the hallmarks of this family. When over-expressed in cells, Bax also causes death. Under non-apoptotic conditions, the localization of Bax is mostly cytosolic, with some mitochondria localization. However, Bax induces apoptosis in response to a variety of death signals, and the association of Bax with mitochondria is linked to the release of cytochrome c, and other death-inducers, from mitochondrial reserves. Data indicate that Bax possesses an intrinsic propensity and capability of membrane pore formation.
Despite advances in understanding the physiology and pathophysiology of cell renewal and apoptosis, there remain many unanswered questions. There is a need for an efficient and inexpensive method of producing and purifying large quantities of Bax protein and Bax protein products. These needs and other needs are satisfied by the present invention.